Cancer treatments such as high-dose chemotherapy and radiation destroy hematopoietic cells in the bone marrow, leaving the patients severely depleted of neutrophils and platelets. After such treatments, patients often spend several weeks in intensive care due to infections and fever resulting from neutropenia. Thrombocytopenia leads to reduced clotting and bleeding disorders requiring platelet transfusions. Lack of neutrophils and platelets is the leading cause of morbidity and mortality following these cancer treatments, and contributes to the high cost of cancer therapy.
Several methods are directed towards restoring the patient's immune system after therapy. Hematopoietic growth factors are administered after therapy to stimulate remaining stem cells to proliferate and differentiate into mature infection fighting cells. Although hematopoietic growth factors can shorten the total period of neutropenia, there remains a critical 10-15 day period immediately following therapy when the patient is severely neutropenic and thus infection prone. Even with growth factor stimulation, 10 to 15 days are required for the patient's stem cells to proliferate and progress through the various stages of differentiation leading to mature neutrophils. Megakaryocyte and platelet recovery requires an even longer time, generally greater than 15 days. Thus growth factor treatment leaves a gap during which the patient is deficient in infection fighting cells and blood clotting ability.
Post-therapy bone marrow transplantation can also ameliorate neutropenia after about 10-15 days. Since allogenic bone marrow transplantation is often complicated by Graft versus Host Disease, autologous bone marrow transplantation is preferred whenever practical. Bone marrow is harvested from the patient prior to therapy, frozen, and then thawed and transplanted back into the patient after therapy. Autologous bone marrow transplantation carries the risk that the transplanted bone marrow may harbor tumor cells. In any event, bone marrow does not contain sufficient mature neutrophils or their immediate precursors to restore the patient's immunity during the critical 10-15 day period after therapy.
A phenomenon known as "mobilization" has also been exploited to harvest greater numbers of stem/progenitor cells from peripheral blood to treat neutropenia. Mobilization occurs as a result of either chemotherapy, or administration of hematopoietic growth factors, or both. It is believed that hematopoietic stem cells in the bone marrow are "mobilized" into the peripheral blood stream as a natural result of the recovery of myelosuppressed bone marrow or in response to relatively large doses of hematopoietic growth factors. Growth factors used for mobilization include interleukin-3 (IL-3), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stem-cell factor (SCF) and a recombinant fusion protein having the active moieties of both IL-3 and GM-CSF (Brandt, S J, et al., N Eng J Med 318:169, 1988; Crawford, J, et al., N Eng J Med 325:164, 1991; Neidhart, J, et al., J Clin Oncol 7:1685, 1989). Mobilized peripheral blood stem cells are harvested after chemotherapy or growth factor treatment, and then reinfused into the patient following the next round of high dose chemotherapy or radiation. The reinfused stem cells then proliferate and differentiate in vivo, eventually to restore the patient's neutrophil and platelet population.
Combinations of the above approaches have succeeded in reducing the neutropenic period to about 9 days.
In order to obtain cells to treat patients during the early neutropenic period, differentiated hematopoietic cells were generated in vitro in the laboratory of the present inventors (Smith, S. L., et al., Experimental Hematology 21:870-877, 1993). Committed precursors of neutrophils were successfully generated in vitro using fetal bovine and horse serum-containing media. However, the potential for therapeutic use of the cells would be greatly enhanced if the cells were grown without animal sera or animal proteins in the culture medium.
Traditionally, animal serum supplementation was relied upon as a source for protein and growth factors in culture media formulations. Researchers have long sought media formulations free of animal proteins for the growth of therapeutic cells because of the potential for life-threatening immune reactions raised by infusion of foreign proteins. However, animal sera, and in particular fetal bovine sera, contain many unknown growth factors, certain factors being more or less important for each cell type. Even if every factor in fetal bovine serum were identified and chemically synthesized, it would still be a matter of trial and error and much experimentation to discover which factors promote the proliferation and differentiation of each cell type. In spite of the difficulty, researchers have succeeded in formulating serum-free media in which certain hematopoietic cells may be grown.
Serum-free media formulations containing bovine serum albumin and various hematopoietic growth factors were shown to promote the proliferation of murine bone marrow cells (Ponting, I. L. O., et al., Growth Factors 4:165-173, 1991; Merchauv, S., et al., Internatl J Cell Cloning 2:356-367, 1984;).
Thirteen different combinations of serum-free media proved disappointing as replacements for serum containing medium for the in vitro culture of human hematopoietic progenitors (Wu, Z-H., et al., Pathology 22:145-148, 1990). The effects of serum-free medium on the growth of leukemic cells were reported (Da, W. M., et al., Brit J Haematology 78:42-47, 1991). The growth of erythropoietic cells in serum-free medium was also reported (Lansdorp, P. M., et al., J Exp Med 175:1501-1509, 1992). All of the above serum-free formulations contained animal protein in the form of bovine serum albumin.
There remains a need for a serum-free, animal protein-free medium formulation which supports the growth and differentiation of human neutrophil and megakaryocyte precursors. The resulting cell suspension would then be suitable for infusion into a patient to restore infection fighting cells and thrombocytes during the critical period immediately following cancer therapy.